Generation of nitric oxide (NO) from L-arginine in mammalian tissues is becoming an area of intense investigation. Diverse physiologic roles are thus being demonstrated or postulated suggesting that NO is an important effector molecule in vascular responses, platelet activity, neurotransmission and inflammation. The most clearly established role for NO from inflammatory/immunologically competent cells is the cytotoxic effect of activated macrophages. Central to the present proposal is the virtually unstudied generation of NO from mast cells and eosinophils, both inflammatory cells implicated in allergic disorders, arthritis, neurogenic inflammation and the underlying pulmonary inflammation of asthma. In contrast to the homeostatic roles of NO, its exaggerated release by these activated inflammatory cells may contribute to tahe pathophysiologic symptoms of certain inflammatory/allergic diseases. Using established in vitro mast cell and eosinophil cell culture models, the l-arginine/NO pathway will be explored in activated cells. Proposed specific aims focus on experiments designed to investigate generation of NO following cell stimulation via various signal transduction pathways. Mast cells will be stimulated with IgE-anti-dinitrophenol BSA, phorbol myristate acetate alone and with ionophore A23187, Substance P, gamma-interferon, tumor necrosis factor-alpha and lipopolysaccharide. Eosinophils will be stimulated with gamma-interferon, tumor necrosis factor-alpha, lipopolysaccharide, the bacterial cell wall peptide fMLP, platelet activating factor, phorbol myristate acetate and Substance P. Thus, cells will be stimulated via immunologic pathways, peptide receptor stimulation and protein kinase C activation. Modulatory roles of superoxide and superoxide dismutase on No release will be explored. Isoform-selective pharmacologic inhibitors of NO synthase will be used to describe the type(s) of NO synthase isoforms present in mast cells and eosinophils. Successful completion of these investigations may yield results useful in the future development of new therapeutic approaches to the treatment of inflammatory diseases without compromising homeostatic vascular pressor mechanisms of NO.